Transcriptomic and Metabolomic Differences Between Two Saposhnikovia divaricata (Turcz.) Schischk Phenotypes With Single- and Double-Headed Roots

Saposhnikovia divaricata is derived from the dried roots of Saposhnikovia divaricata (Turcz.) Schischk and used as a Chinese herbal medicine for treating respiratory, immune, and nervous system diseases. The continuously increasing market demand for traditional Chinese medicine requires the commercial cultivation of Saposhnikovia divaricata using standardized methods and high yielding genotypes, such as double-headed root plants, for achieving consistent quality and a reliable supply. In this study, we aimed to identify the quantitative differences in chromone, a precursor of flavonoid biosynthesis, between plants with single- and double-headed roots using high-performance liquid chromatography and further explore the two phenotypes at the transcriptomic and metabolomic levels. Our results showed that the chromone content was significantly higher in plants with double-headed roots than in those with single-headed roots. Transcriptomic analysis revealed six significantly differentially expressed genes between the two phenotypes, including five key genes in the flavonoid biosynthesis pathway (4-coumarate-CoA ligase, chalcone synthase 1, vinorine synthase, chalcone-flavonone isomerase 1, and flavanone 3 beta-hydroxylase) and one key gene in the abscisic acid biosynthetic pathway (zeaxanthin epoxidase). Moreover, metabolomic analysis showed that the 126 differentially expressed metabolites were mainly enriched in the biosynthesis of secondary metabolites and phytohormones. Overall, our results suggest that plants with double-headed roots have higher medicinal value than those with single-headed roots, probably due to differences in various biosynthetic pathways. These data might help select the genotypes with superior yield and therapeutic properties.

QUANTITATIVE CONTENT OF THE MAIN ACTIVE SUBSTANCES IN THE ROOTS OF THE NATURAL AND IN-TRODUCED PLANT SAPOSHNIKOVIA DIVARICATE (TURCZ.) SCHISCHK.

A HPLC-UV method was developed for the quantitative analysis of chromones in the roots of Saposhnikovia divaricata (Turcz.) Schischk. The optimal parameters of chromones extraction (two-fold ultrasonic extraction with a duration of 40 min with 50% ethyl alcohol at a raw material-extractant ratio of 1:10) were selected. The validation analysis showed that the proposed method is characterized by satisfactory metrological parameters. The method was used for the analysis of raw materials of wild, introduced and commercial samples of S. divaricata. In this work, for the first time in the roots of S. divaricata from natural populations of Buryatia, Trans-Baikal Territory, Mongolia and in the introduced samples, the HPLC-UV method was used to determine the quantitative content of the main active substances: prim-O-glucosylcimifugine (0.13–5.22 mg/g), cimifugine (0.01– 1.82 mg/g) and 4’-O-β-D-glucosyl-5-O-methylvisamminol (0.98–3.25 mg/g). The total content of chromones varies depending on the place of collection and the phase of vegetation. The content of chromones in two samples of roots from introduced S. divaricatae, collected in the first year of introduction in different climatic conditions, was established.

Metabolomic Investigation of Synergistic Mechanism for Fangfeng Extract Preventing LPS Induced Neuroinflammation in BV-2 Microglia Cells

The root of Saposhnikovia divaricata (Fangfeng) is commonly used in traditional Chinese medicine (TCM) for headache and neuroinflammation-related disease treatment. The mRNA expression of IL-6 and IL-1β were significantly inhibited after Fangfeng extract (FFE) treatment in LPS-induced BV-2 cells. Metatolome profiling indicated that dopamine, palmitic acid, corticosterone, and eicosapentaenoic acid metabolites could be regulated by FFE for LPS stimulated inflammation responses in BV-2 cells. The disturbed metabolic pathways include caffeine metabolism, mannose type O-glycan biosynthesis, arachidonic acid metabolism, and steroid biosynthesis. This study will enable us to identify potential protein targets and metabolite intermediates for FFE exerting its protective function in BV-2 cells, and it also provided a potential application of Fangfeng in neuroinflammation-related disease treatment.

Morphological and molecular identification of two new Alternaria species (Ascomycota, Pleosporaceae) in section Radicina from China

The fungal genus Alternaria was distributed widely and found in different habitats such as plant or indoor environment. During an investigation into this genus in China, two new Alternaria species, Alternaria vulgarae and A. divaricatae were respectively isolated from diseased leaves of Foeniculum vulgare and Saposhnikovia divaricata, which both belonged to Umbelliferae. Phylogenetically, they were determined as new species belonging in the section Radicina of Alternaria based on the combined four gene fragments of ITS, TEF1, GAPDH and RPB2. Morphologically, the two species were illustrated and compared with other relevant Alternaria species in section Radicina.

A study on the attenuation of ATO cardiotoxicity by SOG though Nrf2/ARE pathway

Arsenic trioxide (ATO, Trisenox) is an ancient Chinese medicine which have good clinical effects for acute promyelocytic leukemia (APL). However, ATO has serious cardiotoxicity which has restricted its widely applied in clinic. Saposhnikovia divaricata (Trucz.) Schischk was an antidote to ATO in Chinese medical book. Sec-O-Glucosylhamaudol (SOG) is one of its four main active ingredients. So we explore if SOG can detoxify the ATO’s cardiotoxicity through the Nuclear factor erythroid 2-related factor 2(Nrf2) signal pathway and apoptosis pathway. We added 10 μM ATO and 10, 50, 100 μM SOG in Rat cardiac myocyte-like H9c2 cells. The results indicate SOG can active Nrf2 signal pathway, improve Nrf2 phosphorylation levels, promote the expression of Nrf2, HO-1, NQO1 gene and protein, increase the contents of antioxidant enzyme SOD, CAT and GSH-PX, reduce the ROS levels, increase cell activity. By testing the apoptosis-related protein Bax, Bcl-2 and caspase-3, also proved that 10 μM SOG can improve ATO-induced cell apoptosis. So SOG can plays a role in detoxifying ATO cardiac toxicity.

The Species Identification in Traditional Herbal Patent Medicine, Wuhu San, Based on Shotgun Metabarcoding

Traditional herbal patent medicine typically consists of multiple ingredients, making it challenging to supervise contamination by impurities and the improper use of raw materials. This study employed shotgun metabarcoding for the species identification of biological ingredients in traditional herbal patent medicine, Wuhu San. The five prescribed herbal materials found in Wuhu San were collected, and their reference sequences were obtained by traditional DNA barcoding using Sanger sequencing. Two lab-made and three commercial Wuhu San samples were collected, and a total of 37.14 Gb of shotgun sequencing data was obtained for these five samples using the Illumina sequencing platform. A total of 1,421,013 paired-end reads were enriched for the Internal Transcribed Spacer 2 (ITS2), psbA and trnH intergenic spacer region (psbA-trnH), maturase k (matK), and ribulose-1, 5-bisphosphate carboxylase (rbcL) regions. Furthermore, 80, 11, 9, and 8 operational taxonomic units were obtained for the ITS2, psbA-trnH, matK, and rbcL regions, respectively, after metagenomic assembly, annotation, and chimeric detection. In the two lab-made mock samples, all labeled ingredients in the Wuhu San prescription were successfully detected, and the positive control, Panax quinquefolius L., was detected in the HSZY172 mock sample. Three species, namely Angelica sinensis (Oliv.) Diels, Saposhnikovia divaricata (Turcz. ex Ledeb.) Schischk., and Carthamus tinctorius L., belonging to three labeled ingredients, Angelicae Sinensis Radix (Danggui), Saposhnikoviae Radix (Fangfeng), and Carthami Flos (Honghua), were detected in the three commercial samples. Angelica dahurica (Hoffm.) Benth. & Hook. f. ex Franch. & Sav., the original Angelicae Dahuricae Radix (Baizhi) species, was only detected in WHS003. Arisaema erubescens (Wall.) Schott, Arisaema heterophyllum Blume, or Arisaema amurense Maxim., the original Arisaematis Rhizoma (Tiannanxing) species, were not detected in any of the commercial samples, which could be attributed to the fact that this medicinal material underwent extensive processing. In addition, the Saposhnikovia divaricata adulterant was detected in all the commercial samples, while 24 fungal genera, including Aspergillus, were identified in both the lab-made and commercial samples. This study showed that shotgun metabarcoding provided alternative strategy and technical means for identifying prescribed ingredients in traditional herbal patent medicine and displayed the potential to effectively complement traditional methods.